There are over one million people in the United States infected with HIV, and due to the introduction of highly active antiretroviral therapy, HIV infection has become a chronic disease frequently co-existing with chronic use of prescribed, experimental, and illicit drugs, including marijuana. In addition to being the most potent psychoactive component of marijuana, 9-tetrahydrocannabinol (THC) has many other biological actions including modulation of the immune response, neuronal plasticity and food intake. These actions are mediated by interactions with at least three specific receptors (CB1R, CB2R and GPR55) that are expressed in the central nervous system (CNS). However, there are currently no data on the effects of HIV-infection on expression levels and signaling of cannabinoid receptors, or on the effects of cannabinoid receptor activation on the progression of HIV-infection. In our recent studies we observed that long-term THC treatment in SIV- infected rhesus macaques was found to have beneficial effects like decreasing viral load and decreasing early mortality. To test if these effects were due to changes in cannabinoid receptor expression, CB1R and CB2R protein levels were determined in hippocampal extracts. Surprisingly, SIV-infection has differential effects, CB1R levels being reduced to 42 1 8 %, whereas CB2R levels were increased with 53 1 10%. To elucidate the cellular mechanisms underlying these changes, this project will focus on the effects of HIV-1 on the cannabinoid receptors in microglial cells, as these are main immune cells at this level, playing major roles in HIV1-associated dementia (HAD), which affects 10-20 % of AIDS patients. In preliminary experiments we found that both CB1R and CB2R are expressed in human primary microglial cells, but only CB2R levels were upregulated four days post HIV-1 infection. Despite this increase, the effects of CB2R stimulation on the cAMP levels were blocked by HIV-1 infection. In contrast, CB1R activation did not change cAMP levels, but MAPK activation by this receptor was apparent only after HIV-1 infection. In addition, we found that the cellular levels of the scaffold protein 2-arrestin were upregulated after HIV-1 infection, possibly contributing to the observed changes in the cannabinoid signaling. Based on these observations we hypothesize that HIV-1 infection changes the expression levels and signaling of cannabinoid receptors in microglial cells, at least in part by altering 2-arrestin cellular levels. In the Specific aim 1 we will test if HIV-1 infection specifically increases the CB2R levels in human microglial cells, and in the Specific Aim 2 we will test if HIV-1 infection alters the CB1R- and CB2R-mediated effects on cAMP and MAPK levels through alterations in the cellular 2-arrestin levels. When completed, the results from these studies will show for the first time that HIV-1 infection modulates the expression and signaling of cannabinoid receptors in human microglial cells and these data will also provide a clearer understanding of the cellular mechanisms contributing to HIV disease progression in CNS and possibly identify new therapeutics for HIV infection by identifying new drug targets for the treatment of HAD. PUBLIC HEALTH RELEVANCE: The number of microglial cells, the main immunological cell type present in CNS, are increasing during inflammatory processes, including HIV infection, and it has been proposed that the activation of these cells contribute to HIV1-associated dementia (HAD), which has an incidence of 10-20% in AIDS patients. The function of the cannabinoid receptors, proteins which are present in these cells and recognize the main active compound from marijuana, as well as other compounds which are naturally present in the body, has not been investigated in AIDS, although there are indications that drugs interfering with the activity of the cannabinoid receptors may produce positive outcomes in the inflammatory diseases. This application will characterize the regulation of cannabinoid receptor number and function in control and HIV1 infected microglial cells, providing foundation for development of new treatments in AIDS, particularly in HAD.